In order to allow proteins assuming a principal role in the biological life functions to orderly exert their functions in the cellular world, post-translation modifications including glycosylation play an extremely important role. In recent years, it has been gradually revealed that most in vivo proteins undergo modifications with sugar chains and that those sugar chains attached to proteins play important roles in various aspects of biological phenomena such as viral infection, protozoan parasitism and infection, binding of toxins, binding of hormones, fertilization, development and differentiation, protein stability, cancer cell metastasis, and apotosis.
It is known that even those proteins having an identical amino acid sequence (proteins with the same name) are modified with a wide variety of sugar chains and the structures of these sugar chains vary depending on the condition of the protein-producing cells.
The relationships between such changes in sugar chains and diseases have also been gradually elucidated and the findings have been applied to, for example, such methods of identifying hepatoma by fraction measurement of α-fetoprotein (AFP) sugar chains as disclosed in Patent Document 1 and Non-patent Document 1, such methods of identifying prostate adenocarcinoma by fraction measurement of prostate-specific antigen (PSA) sugar chains as disclosed in Patent Document 2, and such methods of identifying adenocarcinoma by fraction measurement of carcinoembryonic antigen (CEA) sugar chains as disclosed in Non Patent Documents 2 and 3.
For specific detection of a sugar chain on a glycoprotein, proteins called lectins that are capable of specifically recognizing, binding to and cross-linking a sugar chain are widely utilized. This is because it is very difficult to prepare an antigen against a sugar chain and such an antigen is hardly available.
Lectins are inexpensive and available in a large amount. In addition, lectins show excellent protein stability and can thus be stored over a prolonged period. However, lectins have such drawbacks of having lower binding activity and specificity than antibodies.
For example, N-acetylgalactosamine is known as a main binding sugar chain in Wisteria floribunda lectin (WFA); however, since N-acetylgalactosamine shows slight binding with galactose, it may non-specifically react with a galactose residue if present in a reaction system.
As a method of simply and quantitatively analyzing a sugar chain on a particular protein using a lectin having such a property, a sandwich assay using an antibody against a protein and a lectin is exemplified.
However, although this technique is effective when the subject protein is purified to a certain extent, since it shows markedly reduced performance in terms of sensitivity and quantitative capacity in a system that contains a large amount of contaminants, such as glycoproteins and glycolipids that are not measurement subjects and included in blood, urine and the like to be used as a sample in ordinary disease diagnosis, it is very difficult to perform an analysis using this technique. Accordingly, this technique is utilized only in serum diagnosis and the like of limited items in which the serum concentration of subject protein is extremely high (about several μg/mL).
As measures for reducing the effects of such contaminants, the use of a blocking agent which inhibits adsorption of serum contaminants to the surface of an antibody-immobilized support (Patent Document 3), the addition of an adsorbent which allows non-specific substances to be adsorbed in advance (Patent Documents 4 and 5) and the use of a washing solution capable of efficiently removing molecules adsorbing to a support (Patent Document 3) have been examined.
Here, as blocking agents, for example, bovine serum albumin (BSA) and casein are known, and synthetic macromolecular materials are also used in some cases, An absorbent for non-specific substances is a molecular which is capable of binding with molecules that inhibit antigen-antibody reaction and cause noise generation and removing such molecules from a reaction system. As such an absorbent, macromolecules and sugar chain complexes such as glycosaminoglycan and heparin may be used. As for a washing solution, the composition thereof has been examined and there is also a report of a case where the effects of salt strength and various surfactants are investigated (Patent Document 3).
However, it is an extremely rare case where these background-suppressing measures show a drastic effect in quantitative analysis using a lectin for detection, and there is also a problem that the search and examination of subject-suitable blocking agent require tremendous man-hours.